The invention pertains to a xcex2-(1,3) exoglucanase gene of Coniothyrium minitans. 
The plant cell wall provides stability, protects against pathogens, and influences the growth and development of the plant cell, among other functions. Structurally, the plant cell wall consists of a primary and a secondary wall, both containing cellulose microfibrils embedded in a matrix of carbohydrates (specifically polysaccharides), structural glycoproteins, enzymes, and other components. Carbohydrate polymers have been well characterized and play a primary role in maintaining the structural rigidity of the plant cell wall. In this regard, the plant cell wall sequesters significant amounts of metabolically inactive polysaccharides from among the following classes:
i) celluloses (insoluble fibrils of xcex2-(1,4) glucans);
ii) hemi-celluloses (non-cellulosic polysaccharides which include xcex2-(1,3) glucans, xcex2-(1,3))(1,4) glucans, mannans, and xylans); and
iii) lignin (a polyphenolic compound) (Thomson, 1993).
The xcex2-glucans are polymers of glucose molecules formed by xcex2-links between the glucose molecules. The links may be xcex2-(1,4), xcex2-(1,3), or xcex2-(1,6) or a mixture of those in such polymers. xcex2-glucans are ubiquitous in the natural flora. Many classes of xcex2-glucan polymers exist, and their chemical structure, physiological function, and predominance differ among plant and fungal species.
A. Cellulosic xcex2-Glucans
Cellulosic xcex2-(1,4) glucans are polymeric chains formed by successive glucose monomers covalently joined by xcex2-(1,4) glucan linkages. These xcex2-(1,4) glucan chains associate In bundles to form rigid, insoluble microfibrils which may contain up to several hundred cellulosic polymers (Beguin and Aubert, 1994). The tensile strength of such cellulose microfibrils in the plant cell wall selves to confer rigidity to plant structures. Further, cellulosic components, together with other polymeric compounds in the plant cell wall, demonstrate a protective role by acting as a barrier to various phytopathogens.
B. Non-cellulosic xcex2-Glucans
While cellulosic polymers are ubiquitous in the cell walls of diverse plant species, non-cellulosic glucans (xcex2-(1,3) glucans and xcex2-(1,3)(1,4) glucans) are typically present in the cell walls of some monocotyledonous plant families, such as the Poaceae (Gramineae) (Chesson et al., 1995). In fungi, non-cellulosic xcex2-(1,3) glucans are predominant in the cell wall, notably providing structural resilience (Borgia and Dodge, 1992). In addition to providing structural stability to the fungal cell wall, xcex2-(1,3) glucans serve as carbohydrate reserves in nutritionally-depleted growth environments (Copa-Patino et al., 1989).
The hydrolysis of non-cellulosic xcex2-glucans by xcex2-glucanase enzymes is of great significance to plant-mycopathogen interactions, fungal cell wall architecture, and forage feed digestion in ruminants (Umemoto et al., 1997; Vasquez-Garciduenas et al., 1998; Chen et al., 1997). Such enzymes have been classified into different families according to their origin (plant, fungal, or microbial), substrate specificity, and function (Table 1). Different non-cellulosic xcex2-glucanases thus have distinct substrates and modes of hydrolytic action, to the extent that plant, fungal, and microbial non-cellulosic xcex2-glucanases each belong to specific families with conserved sequence and functional properties.
i) Non-Cellulosic xcex2-Glucanases in Plants
In plants, non-cellulosic glucanases may be classified as either xcex2-(1,3) endoglucanases (laminarinases) or xcex2-(1,3)(1,4) glucanases (mixed linkage glucanases or lichenases) according to substrate specificity and function (Table 1). xcex2-(1,3) endoglucanases (EC 3.2.1.39) hydrolyze successive xcex2-(1,3) glucan (laminarin) chains in an endoglucanase manner (i.e. random digestion within the polymeric chain), whereas xcex2-(1,3)(1,4) glucanases (EC 3.2.1.73) specifically degrade mixed-linkage glucans (non-cellulosic glucans containing glycosidic xcex2-(1,3) and xcex2-(1,4) linkages such as lichenan) by hydrolyzing a xcex2-(1,4) linkage adjacent to a xcex2-(1,3) linkage in the same manner (Hoj and Fincher, 1995).
In addition to targeting different substrates, xcex2-(1,3) endoglucanases and xcex2-(1,3)(1,4) glucanases are distinct functionally. xcex2-(1,3) endoglucanases appear to comprise a large family of pathogenesis-related proteins produced by plants during infection by pathogens. During the plant-pathogen interaction between soybean plants (Glycine max) and the fungal pathogen Phytophthora megaspora f. sp. glycinea, soybean xcex2-(1,3) endoglucanases are able to digest the fungal cell walls (Umemoto et al., 1997). The liberated fungal xcex2-(1,3) oligoglucans subsequently bind a xcex2-oligoglucan receptor in the plant cell membrane, initiating a signal transduction event, and ultimately stimulating plant defense responses such as phytoalexin accumulation. xcex2-(1,3) endoglucanases thus appear to weaken and degrade fungal cell walls, while liberating elicitor compounds (such as xcex2-oligoglucan) in order to upregulate plant defense responses.
In comparison, xcex2-(1,3)(1,4) glucanases may play an important role in nutrient mobilization during seed germination in some plant species. During barley (Hordeum vulgare) seed germination, the xcex2-(1,3)(1,4) glucanases degrade the xcex2-(1,3)(1,4) glucan-rich cell wall in the seed endosperm, allowing the diffusion of amylases and proteases into starch and protein stores in the endosperm compartment (Hoj and Fincher, 1995).
Although xcex2-(1,3) endoglucanases thus differ functionally from xcex2-(1,3)(1,4) glucanases, these glucanase types in plants are structurally conserved, appearing to originate from a common ancestor (Hoj and Fincher, 1995).
ii) Non-Cellulosic xcex2-Glucanases in Fungi
In comparison to xcex2-(1,3) endoglucanases and xcex2-(1,3)(1,4) glucanases in plants, fungal glucanases differ in both sequence and function (Table 1). In fungi, non-cellulosic glucanases consist of the following classes: xcex2-(1,3) exoglucanase (EC 3.2.1.58); xcex2-(1,3) endoglucanase (EC 3.2.1.39); xcex2-(1,3)(1,4) endoglucanase (EC 3.2.1.73); and xcex2-(1,3)/(1,3)(1,4) glucanase (EC 3.2.1.6). Fungal xcex2-(1,3) exoglucanases are quintessential enzymes in mycoparasitism. Mycoparasites, such as Trichoderma hazarium, rely on xcex2-(1,3) exoglucanases to hydrolyze the cell wall of various fungal phytopathogens, thus liberating nutritionally available oligoglucans for absorption and metabolism (Vasquez-Garciduenas et al., 1998). Further, fungal xcex2-(1,3) exoglucanases have been implicated in the autolysis of fungal cell walls in nutritionally-depleted environments (Copa-Patino et al., 1989; Stahmann et al., 1993). In addition, xcex2-(1,3) exoglucanases may have a morphogenic role in fungal growth and differentiation (Peberdy, 1990).
The prevalence of xcex2-(1,3)(1,4) endoglucanases in fungi has yet to be confirmed. To date, few of these have been cloned, with the pioneering example being a mixed-linkage glucanase from the ruminal anaerobic fungus Orpinomyces (licA) (Chen et al., 1997). Such mixed-linkage glucanases from ruminal organisms are presumably produced to improve the digestibility of non-cellulosic xcex2-glucans from fibrous forage feed.
iii) Non-Cellulosic xcex2-Glucanases in Bacteria
In bacteria, non-cellulosic glucanases consist of xcex2-(1,3)(1,4) glucanases (EC 3.2.1.73), which are specific for the substrate, xcex2-(1,3)(1,4) glucan (Table 1). Examples of such microbial glucanases include enzymes from ruminal and non-ruminal microbial species (e.g. Fibrobacter succinogenes and Bacillus subtilis respectively) (Teather and Erfle, 1990; Wolf et al., 1995).
iv) Non-Cellulosic xcex2-Glucanases in Lower Animalia
A metazoan xcex2-(1,3) endoglucanase from the sea urchin Strongylocentrotus purpuratus has been characterized, apparently having a bacterial origin (Bachman and McClay, 1996). Its presence in sea urchin eggs implies that the enzyme may have a glucanolytic function in embryogenesis. Although the role of xcex2-glucanases in metazoans remains obscure, the mere presence of xcex2-glucanases in natural flora and fauna demonstrates the significance of glucanohydrolytic enzymes among a diverse spectrum of biological systems.
v) Applications for Non-Cellulosic xcex2-Glucanases
The developing interest in non-cellulosic xcex2-glucanases ranges from the elucidation of their basic enzymatic action to their numerous industrial applications. Although they have yet to be used extensively in commercial applications, non-cellulosic xcex2-glucanases have already been used to hydrolyze and clarify barley xcex2-glucan in brewing processes (Bamforth, 1980).
Specific interest in non-cellulosic xcex2-glucanases has stemmed from the production of plant xcex2-(1,3) endoglucanases in response to fungal infection. Although such enzymes participate in pathogen responses with some degree of efficacy, the incorporation of a xcex2-(1,3) glucanase with superior hydrolytic activity into a pathogen response regimen may improve plant resistance to fungi. In this regard, compatible xcex2-(1,3) glucanase genes may be incorporated into a transgenic plant line under the transcriptional regulation of a pathogen-responsive promoter.
Furthermore, the development of non-cellulosic xcex2-(1,3)(1,4) glucanases in ruminant microbial technology may increase the efficiency by which non-cellulosic fiber (such as barley xcex2-glucan) is digested. Hence, compatible glucanolytic genes may be incorporated into ruminant microbial or fungal species to improve fiber digestion and nutritive carbohydrate availability from forage feed. Acquisition and characterization of novel non-cellulosic xcex2-glucanases are thus essential towards the use of glucanase genes in various transgenic applications, and the study of the functional flexibility of xcex2-glucanase enzymes.
Coniothyrium minitans is a higher eukaryotic fungal mycoparasite which is ubiquitous in soil and non-pathogenic to plants and animals. C. minitans exhibits marked xcex2-glucanolytic properties, indicating potential for its development in biotechnological and transgenic applications. C. minitans culture supernatants have been previously shown to be effective in hydrolyzing fungal residue of the phytopathogenic organism Sclerotinia sclerotiorum (Lib.) de Bary by cooperative activity of xcex2-(1,3) exo- and xcex2-(1,3) endoglucanases (Jones et al., 1974). Additionally, the production of extracellular xcex2-glucanohydrolases was induced by the presence of xcex2-glucan-rich complex carbohydrate sources found in fungal cell walls (International Publication No. WO 99/02662 to Huang et al.). C. minitans xcex2-glucanases have also been implicated in S. sclerotiorum hyphal and sclerotial invasion, penetration, and degradation (Huang and Hoes, 1976; Huang and Kokko, 1987; Huang and Kokko, 1988).
Fungal non-cellulosic xcex2-glucanases are rare enzymes for which only a few sequences are presently known and available for comparison and functional extrapolation to their homologous counterparts. Isolation and characterization of novel fungal xcex2-glucanases will consolidate functional studies based on gene sequence homologies. Moreover, the discovery of novel glucanolytic sequences will actively contribute to an expanding database containing potential sequences for various biotechnological applications.
The invention provides a novel xcex2-(1,3) exoglucanase gene (denoted herein as cbeg1) of the soil-borne fungus Coniothyrium minitans. The DNA sequence of the cbeg1 gene and the deduced amino sequence of the encoded xcex2-(1,3) exoglucanase Cbeg1 are depicted in SEQ ID NOS: 1 and 2 respectively. The invention extends to polypeptides possessing xcex2-(1,3) exoglucanase activity, and which comprise amino acid sequences having a length of at least 50 amino acid residues, more preferably at least 100 amino acid residues, more preferably at least 200 amino acid residues, more preferably at least 500 amino acid residues, more preferably at least 600 amino acid residues, more preferably at least 700 amino acid residues, and most preferably at least 750 amino acid residues to the amino acid sequence depicted in SEQ ID NO: 2. In addition, the invention extends to polypeptides possessing xcex2-(1,3) exoglucanase activity, and which comprise amino acid sequences having at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% homology to the amino acid sequence depicted in SEQ ID NO: 2.
The cbeg1 gene is compatible with a eukaryotic heterologous expression system, making it particularly useful for a wide range of industrial applications, such as improvement of plant resistance to fungal phytopathogens or use in non-ruminant and ruminant microbial transgenic strategies to improve feed digestion and nutritive carbohydrate availability from forage feed, whereby Cbeg1 degrades the cell wall from plants, particularly within the Poaceae.
In addition, the high activity of Cbeg1 over broad pH and temperature ranges provides benefits in high temperature industrial applications, such as bleaching of pulp, which require temperatures greater than 37xc2x0 C. Further, Cbeg1 complements degradation initiated by endoglucanases which release oligoglucans, in that xcex2-(1,3) exoglucanase sequentially hydrolyzes xcex2-(1,3) glucan fragments and is required to hydrolyze oligoglucan fragments completely to obtain D-glucose, which can be assimilated. Further, Cbeg1 benefits the plant itself by degrading the cell walls of pathogenic fungi without affecting plant cell walls in dicots, and controlling and stimulating expansion of the cell wall to promote plant growth in monocots and dicots.
Encoded xcex2-(1,3) exoglucanase Cbeg1 is specific for the substrate laminarin, a xcex2-(1,3) glucan with some xcex2-(1,6) linkages, which serves as a carbon reserve polysaccharide in Laminaria and other brown algae (Phaeophyta). Further, Cbeg1 is specific for only laminarin, in that results showed no activity with other substrates tested, such as carboxymethylcellulose, barley xcex2-glucan, lichenan, oat spelt xylan and birchwood xylan. The pH and temperature optima for xcex2-(1,3) exoglucanase Cbeg1 are 6.0 and 57xc2x0 C., respectively. Cbeg1 contains 784 amino acids, and has a predicted isoelectric point (pI) of 6.0 and molecular weight of 83,646 Daltons.
The invention further provides vectors and cells comprising a nucleic acid molecule encoding the cbeg1 gene, and methods for producing xcex2-(1,3) exoglucanase Cbeg1.
As used herein and in the claims, the terms and phrases set out below have the following definitions.
A xe2x80x9cxcex2-(1,3) exoglucanasexe2x80x9d is an enzyme that catalyzes the successive hydrolysis of beta-D-glucose units from the non-reducing ends of 1,3-beta-D-glucans, releasing alpha-glucose. The Official Name for xcex2-(1,3) exoglucanase, as recommended by the International Union of Biochemistry and Molecular Biology (xe2x80x9cIUBMBxe2x80x9d) is xe2x80x9cglucan 1,3-beta-glucosidasexe2x80x9d, and its Enzyme Commission (xe2x80x9cECxe2x80x9d) number is (EC 3.2.1.58). Similarly, a xe2x80x9cpolypeptide having xcex2-(1,3) exoglucanase activityxe2x80x9d is a polypeptide that catalyzes the successive hydrolysis of beta-D-glucose units from the non-reducing ends of 1,3-beta-D-glucans, releasing alpha-glucose.
A xe2x80x9cxcex2-glucanxe2x80x9d is a polymer of glucose molecules formed by xcex2-links between the glucose molecules. The links may be xcex2-(1,4), xcex2-(1,3), or xcex2-(1,6) or a mixture of those in such a polymer.
xe2x80x9cCoding sequencexe2x80x9d means the part of a gene which codes for the amino acid sequence of a protein, or for a functional RNA such as a tRNA or rRNA.
xe2x80x9cComplementxe2x80x9d or xe2x80x9ccomplementary sequencexe2x80x9d means a sequence of nucleotides which forms a hydrogen-bonded duplex with another sequence of nucleotides according to Watson-Crick base-pairing rules. For example, the complementary base sequence for 5xe2x80x2-AAGGCT-3xe2x80x3 is 3xe2x80x2-TTCCGA-5xe2x80x2.
A xe2x80x9cdomainxe2x80x9d of a polypeptide is a portion or region of the polypeptide that forms a structural or functional niche within the remainder of the polypeptide. For example, DNA-binding proteins have DNA-binding domains with specific features such as helix-turn-helix configurations or Zn2+-fingers which enable them to recognize and bind to specific structures or sequences on their target DNA with high specificity and affinity.
xe2x80x9cDownstreamxe2x80x9d means on the 3xe2x80x2 side of any site in DNA or RNA.
xe2x80x9cExpressionxe2x80x9d refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into a protein.
An amino acid sequence that is xe2x80x9cfunctionally equivalentxe2x80x9d to C. minitans Cbeg1 is an amino acid sequence that has been modified by single or multiple amino acid substitutions, by addition and/or deletion of amino acids, or where one or more amino acids have been chemically modified, but which nevertheless retains the xcex2-(1,3) exoglucanase activity of C. minitans Cbeg1.
xe2x80x9cFunctionally equivalentxe2x80x9d nucleotide sequences are those that encode polypeptides having substantially the same biological activity.
Two nucleic acid sequences are xe2x80x9cheterologousxe2x80x9d to one another if the sequences are derived from separate organisms, whether or not such organisms are of different species, as long as the sequences do not naturally occur together in the same arrangement in the same organism.
Two polynucleotides or polypeptides are xe2x80x9chomologousxe2x80x9d or xe2x80x9cidenticalxe2x80x9d if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described herein. Sequence comparisons between two or more polynucleotides or polypeptides are generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window is generally from about 20 to about 200 contiguous nucleotides or contiguous amino acid residues. The xe2x80x9cpercentage of sequence identityxe2x80x9d or xe2x80x9cpercentage of sequence homologyxe2x80x9d for polynucleotides and polypeptides may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and, (c) multiplying the result by 100 to yield the percentage of sequence identity.
Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms, or by inspection. A list providing sources of both commercially available and free software is found in Ausubel et al. (2000). Readily available sequence comparison and multiple sequence alignment algorithms are, respectively, the Basic Local Alignment Search Tool (BLAST) (Altschul e al., 1997) and ClustalW programs. Other suitable programs include GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.). For greater certainty, as used herein and in the claims, xe2x80x9cpercentage of sequence identityxe2x80x9d or xe2x80x9cpercentage of sequence homologyxe2x80x9d of amino acid sequences is determined based on optimal sequence alignments determined in accordance with the default values of the BLASTX program, available as described above.
As discussed in greater detail hereinafter, homology between nucleotide sequences can also be determined by DNA hybridization analysis, wherein the stability of the double-stranded DNA hybrid is dependent on the extent of base pairing that occurs. Conditions of high temperature and/or low salt content reduce the stability of the hybrid, and can be varied to prevent annealing of sequences having less than a selected degree of homology.
xe2x80x9cHemicellulosexe2x80x9d includes glucans (apart from starch), mannans, xylans, arabinans or polyglucuronic or polygalacturonic acid.
xe2x80x9cIsolatedxe2x80x9d means altered xe2x80x9cby the hand of manxe2x80x9d from the natural state. If an xe2x80x9cisolatedxe2x80x9d composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not xe2x80x9cisolatedxe2x80x9d, but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is xe2x80x9cisolatedxe2x80x9d, as the term is employed herein.
xe2x80x9cLaminarinxe2x80x9d means a polymer of xcex2-(1,3) glucan with some xcex2-(1,6) linkages, which serves as a carbon reserve polysaccharide in Laminaria and other brown algae (Phaeophyta).
A xe2x80x9cpolynucleotidexe2x80x9d is a linear sequence of deoxyribonucleotides (in DNA) or ribonucleotides (in RNA) in which the 3xe2x80x2 carbon of the pentose sugar of one nucleotide is linked to the 5xe2x80x2 carbon of the pentose sugar of the adjacent nucleotide via a phosphate group.
A xe2x80x9cpolynucleotide constructxe2x80x9d is a nucleic acid molecule which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed in a manner which would not otherwise exist in nature.
Two DNA sequences are xe2x80x9coperably linkedxe2x80x9d if the nature of the linkage does not interfere with the ability of the sequences to effect their normal functions relative to each other. For instance, a promoter region would be operably linked to a coding sequence if the promoter were capable of effecting transcription of that coding sequence.
A xe2x80x9cpolypeptidexe2x80x9d is a linear polymer of amino acids that are linked by peptide bonds.
xe2x80x9cPromoterxe2x80x9d means a cis-acting DNA sequence, generally 80-120 base pairs long and located upstream of the initiation site of a gene, to which RNA polymerase may bind and initiate correct transcription.
A xe2x80x9crecombinantxe2x80x9d nucleic acid molecule, for instance a recombinant DNA molecule, is a novel nucleic acid sequence formed in vitro through the ligation of two or more nonhomologous DNA molecules (for example a recombinant plasmid containing one or more inserts of foreign DNA cloned into its cloning site or its polylinker).
xe2x80x9cTransformationxe2x80x9d means the directed modification of the genome of a cell by the external application of purified recombinant DNA from another cell of different genotype, leading to its uptake and integration into the subject cell""s genome. In bacteria, the recombinant DNA is not integrated into the bacterial chromosome, but instead replicates autonomously as a plasmid.
A xe2x80x9ctransgenicxe2x80x9d organism, such as a transgenic plant, is an organism into which foreign DNA has been introduced. A xe2x80x9ctransgenic plantxe2x80x9d encompasses all descendants, hybrids, and crosses thereof, whether reproduced sexually or asexually, and which continue to harbour the foreign DNA.
xe2x80x9cUpstreamxe2x80x9d means on the 5xe2x80x2 side of any site in DNA or RNA.
A xe2x80x9cvectorxe2x80x9d is a nucleic acid molecule that is able to replicate autonomously in a host cell and can accept foreign DNA. A vector carries its own origin of replication, one or more unique recognition sites for restriction endonucleases which can be used for the insertion of foreign DNA, and usually selectable markers such as genes coding for antibiotic resistance, and often recognition sequences (e.g. promoter) for the expression of the inserted DNA. Common vectors include plasmid vectors and phage vectors.